Project Summary The human microbiome is gaining widespread attention for its link to host health and disease. This is particularly true of the cystic fibrosis airways, where a complex microbial community is recognized to be the major cause of patient mortality. In addition to Pseudomonas aeruginosa, a recent surge in culture- independent studies has generated a growing list of suspected pathogens and their correlation to disease progression. Yet, our inability to effectively treat these patients remains due to a lack of understanding of the CF lung environment and how specific chemical parameters define the presence or absence of a given bacterial species. In natural environments, such as a marine sediment, defining the effect of environmental chemistry on the microbial community is paramount in fully understanding a given ecosystem. Yet, this approach in a medical microbiology context is seldom used. Here, my overarching goal is to apply standard environmental microbiology principles and methodologies to test the hypothesis that the complex chemistry of the bronchial tree affects its resident microflora on multiple scales. Recently, we discovered a highly significant correlation between a class of microbially-produced metabolites (phenazines), microbiome composition, and lung function decline. In the K99 phase of this award, the overall goal will be to expand on these initial observations, and to broadly investigate the effect of the cystic fibrosis lung environment on its microbiome. Using a cohort of pediatric patients, I will first test that phenazines alter the redox state of iron, which ultimately correlates to a shift in the overall lung microbiome and disease progression. In addition, I will use novel imaging methods to investigate the cellular response of individual species (particularly P. aeruginosa) at the single cell level to changes in environmental chemistry (specifically in response to iron). These studies will reveal the potential of using single cell transcriptional activity as a direct readout of the ambient environment of the airways, and will potentially guide the design of novel therapeutics based on environmental chemistry. Next, we will apply these methods to explanted lung samples in order to better understand the spatial heterogeneity of microbial communities throughout the bronchial tree. With this information in hand, these studies will ultimately be expanded in the R00 phase of the award to include other metabolites and chemical parameters within the respiratory tract, and other sites of infection within the host. Altogether, this systems approach to understanding the ecology of microbial infections will change the way we think about the microbiome and its link to health and disease, and has the potential for development of novel therapeutic strategies.